Among Gram-negative bacteria, nosocomial infections by Klebsiella pneumoniae that are resistant to carbapenems and quinolones are rapidly becoming a global problem. The emerging resistance to imipenem is primarily mediated by KPC beta-lactamases;resistance to quinolones occurs as a result of mutations in the quinolone resistance determining region (QRDR) of gyrA and parC as well as the acquisition of plasmid mediated quinolone resistance genes qnrA, -B, -S, modifying enzymes (aac(6')-Ib-cr) and efflux pumps. The early detection and analysis of carbapenem- and ciprofloxacin- resistant K. pneumoniae will enormously assist clinicians treating patients with serious infections. To that end, a rapid, sensitive, and high-throughput assay to detect, identify, genotype, and determine the antibiotic resistance profile of K. pneumoniae using PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is planned. The specific aims of this proposal are to: 1) compare electrospray ionization mass spectrometry (PCR/ESI-MS) of seven "housekeeping" genes in K. pneumoniae to pulsed field gel electrophopresis and rep-PCR to develop a fast and reproducible method that can applied in any facility;2) determine the sequence of KPC-type beta-lactamases and quinolone resistance determinants in a working collection of 43 K. pneumoniae isolates;3) Integrate the knowledge acquired from 1 and 2 to check simultaneously the ability of PCR/ESI-MS in detection of blaKPC genes and quinolone resistance determinants. By choosing genes that have minimal variability (blaKPC, gyrA, etc.) this approach promises to increase accuracy and to reduce the time needed to detect carbapenem and quinolone resistance. It is hoped that this effort will serve as a model for the development of other "bench to bedside" methods for discovering resistant phenotypes in other enteric bacilli and form the basis for the implementation of real time infection control and diagnostic paradigms. PUBLIC HEALTH RELEVANCE: This application addresses a major unmet need for rapid genotyping and antibiotic resistance profiling of Klebsiella pneumoniae. The timely and correct identification can directly influence lifesaving medical decisions regarding the proper administration of antibiotics to treat infection. We propose to develop a rapid, sensitive, and cost-effective diagnostic method that will be used to detect, identify, genotype, and determine the antibiotic resistance profile of carbapenem- and quinolone-resistant K. pneumoniae. It is our goal that this method, based upon a novel technology, will form the basis for the implementation of real time infection control and diagnostic paradigms.